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foundation for hypotheses and future research concerning the physiological and biochemical roles of each of the retinoid-binding proteins. Acknowledgments The research reported and referred to in this chapter was supportedby National Institutes of Health Grants HL21006and DK05968.

[30] G e l E l e c t r o p h o r e s i s o f C e l l u l a r R e t i n o i c A c i d - B i n d i n g Protein, Cellular Retinol-Binding Protein, and Serum Retinol-Binding Protein B y GEORGES SIEGENTHALER

Introduction Cellular retinoid-binding proteins are involved in the mechanism of action of retinoids.~,2 However, their exact role is not fully understood. The elucidation of retinoid-binding proteins requires the availability of a rapid and sensitive method endowed with a high power of resolution for the detection, discrimination, and characterization of these proteins. Several different retinoid-binding proteins can be detected in a target tissue for vitamin A. The cellular retinoic acid- and retinol-binding proteins (CRABP, CRBP), which are specific for retinoic acid and retinol, respectively, are among the best known. 1-3 The transport and storage of retinoids may also involve nonspecific binding proteins such as fatty acidbinding proteins (FABP). 4,5 Unfortunately, the molecular weights of CRABP, CRBP, and FABP are very close to each other, around 15,000, a fact which renders the gel sieving technique ineffective, in spite of the various specificity tests one can perform with different ligands.

t F. Chytil and D. E. Ong, in "The Retinoids" (M. D. Sporn, A. B. Roberts, and D. S. Goodman, eds.), Vol. 2, p. 89. Academic Press, Orlando, Florida, 1984. 2 M. Mader, D. E. Ong, D. Summerball, and F. Chytil, Nature (London) 335, 733 (1988). 3 p. N. McDonald, and D. E. Ong, J. Biol. Chem. 2£2, 10550 (1987). 4 B. P. Sani, R. D. Allen, C. M. Moorer, and B. W. McGee, Biochem. Biophys. Res. Commun. 147, 25 (1987). 5 F. Fukai, T. Kase, T. Shidotani, T. Nagai, and T. Katayama, Biochem. Biophys. Res. Commun. 147, 899 (1987).

METHODSIN ENZYMOLOGY.VOL. 189

Copyright© 1990by AcademicPress,Inc. All fightsof reproductionin any formreserved.

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RECEPTORS, TRANSPORT, AND BINDING PROTEINS

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FIG. 1. Electrophoresis-immunoblotting analysis with goat anti-human RBP serum of normal serum, chorion, and epithelial extracts of human oral mucosa. Lane 1, Holo- (h) and apo-RBP (a) from human serum; lanes 2 and 3, apo-RBP of chorion and epithelial extracts; lane 4, reconstitution of holo-RBP from apo-RBP of epithelial extracts with 10/zM of retinol. (From Ref. 8 with permission of the publisher.)

Serum retinol-binding protein (RBP), which is supposed to supply retinol from the blood vessels to target cells, might also be an important contaminant in tissue protein extracts 6-8 (Fig. 1) or in the cytosol of cultured hepatic cells. It has been reported that these cells are able to synthesize RBP in addition to CRBP and CRABP. 9 The study of these binding proteins necessitates specific techniques other than gel sieving, charcoaldextran separation, or sucrose density gradient centrifugation. In the serum, holo-RBP (the retinol-RBP complex) is found to represent 98% of the total RBP (holo- and free or apo-RBP).10 Holo-RBP is entirely bound 6 G. Siegenthaler and J.-H. Saurat, J. Invest. Dermatol. 88, 403 (1987). 7 G. Siegenthaler and J.-H. Saurat, Eur. J. Biochem. 166, 209 (1987). s G. Siegenthaler, J. Samson, J.-P. Bernard, G. Fiore-Donno, and J.-H. Saurat, J. Oral Pathol. 17, 106 (1987). 9 j. L. Dixon and D. S. Goodman, J. Cell. Physiol. 130, 14 (1987). to G. Siegenthaler and J.-H. Saurat, Biochem. Biophys. Res. Commun. 143, 418 (1987).

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to transthyretin (TTR), formerly named prealbumin, forming a complex with a molecular weight of 76,000. The tissue level of TTR is lower than the serum level and may not be sufficient to bind RBP completely, so that some RBP could remain free. Moreover, it has been shown that RBP binds retinoic acid (RA) as well as retinol in vitro. 7-~° The resulting RARBP complex does not appear to possess strong binding properties toward TTR because of important structural alterations of the protein core. For these reasons, RA-RBP will remain free in protein extracts. It should be mentioned that the RA-RBP complex migrates faster on polyacrylamide gel electrophoresis (PAGE) than holo-RBP and apo-RBP and just before the CRABP peak. l° The mode of action of RBP involves plasma membrane receptors for RBP, CRBP, and esterifying and hydrolyzing enzymes for retinol and its esters. 1~ Studies of such a model also necessitate specific radiobinding techniques which allow one to measure the levels of holo-RBP and CRBP, whereas an estimation of apo-RBP is possible with PAGE immunoblotting techniques. Sedimentation analysis on sucrose density gradients 12and gel sieving chromatography ~3,~4connected to or independent of high-performance liquid chromatography (HPLC) methods 15,16are currently used for the study of retinoid-binding proteins. However, these methods are extremely time-consuming, and only a limited number of samples can be analyzed simultaneously. In order to diminish the background radioactivity, one has to treat the samples with dextran-coated charcoal after incubation with [3H]retinoids, to remove excess free labeled ligand. If the samples are analyzed by HPLC, previous filtration of the samples through 0.45-/.Lm filters is indispensable, to avoid blocking of the column by unsedimented charcoal particules. Moreover, during analysis with the above technique, the sample is diluted about one-tenth, a fact which decreases considerably the resolution of the various radioactive peaks. In this chapter, we describe procedures for the detection, characterization, and measurement by polyacrylamide gel electrophoresis of CRABP, CRBP, RBP and other retinoid-binding proteins. The results of

ii S. Ottonello, S. Petrucc, and G. Maraini, J. Biol. Chem. 262, 3975 (1987). 12 D. E. Ong and F. Chytil, Proc. Natl. Acad. Sci. U.S.A. 73, 3976 (1976). 13 G. Siegenthaler, J.-H. Saurat, R. Hotz, M. Camenzind, and Y. Mrrot, J. Invest. Dermatol. 86, 42 (1986). 14 G. Siegenthaler, J.-H. Saurat, and M. Ponec, Exp. Cell. Res. 178, 114 (1988). 15 H. E. Shubeita, M. D. Patel, and A. M. McCormick, Arch. Biochem. Biophys. 247, 280 (1986). 16 E. A. Allegretto, M. A. Kelly, C. A. Donaldson, N. Levine, J. W. Pike, and M. R. Haussler, Biochem. Biophys. Res. Commun. 116, 75 (1983).

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F/o. 2. Electrophoresis analysis of labeled retinoid-binding proteins from human skin. (A) Epidermis extract incubated with [3H]retinoic acid in the absence (O) or presence (0) of excess retinoic acid. (B) Dermis extract incubated with [3H]retinol in the absence (O) or presence (0) of retinol. (From Ref. 7 with permission of the publisher.)

t y p i c a l e x p e r i m e n t s f o r t i s s u e p r o t e i n e x t r a c t o r c y t o s o l a r e p r e s e n t e d in F i g s . 2 a n d 3.

Separation of Retinoid-Binding Proteins by Electrophoresis

Principle. The protein extracts arc incubated with the various labeled retinoids before being subjected to vertical slab P A G E under nondcnaturing conditions. The proteins are then separated according to their different clcctrophoretic mobilities in the gel, as a consequence of their net charge and molecular weight. After clectrophoresis, the gel is divided into lanes and cut into 2-ram bands which are counted for the determination of the radioactivity profile. The technique of P A G E allows the separation of C R A B P , CRBP, R B P and other rctinoid-binding proteins in one run. Moreover, 10 different samples can be analyzed in the same gel. During gel clectrophoresis, there is practically no dissociation of the ligands from the binding proteins, whereas the complexes of retinoidbinding proteins with other proteins dissociate readily. For example, the

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PAGE ANALYSISOF CRABP, CRBP, AND RBP

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10 20 30 40 50 30 40 50 number of band (2 mini number of band 12mm} FIG. 3. Electrophoresis analysis of CRABP and CRBP from cultured differentiated human keratinocytes. (A) Cytosol incubated with pH]retinoic acid in the absence ([2) or presence of excess retinoic acid (×) or retinol (e). (B) Cytosol incubated with [3H]retinolin the absence (D) or presence of excess retinol (X) or retinoic acid (11). (From Ref. 14 with permission of the publisher.) 10

20

h o l o - R B P - T T R complex is totally dissociated into holo-RBP and TTR. This p h e n o m e n o n might explain the higher level of CRABP found with the P A G E technique by comparison with gel sieving, which reveals the presence of high molecular weight retinoid acid-binding proteins besides CRABP. TM It is not necessary to r e m o v e excess labeled retinoid, which either migrates with the front of the gel in a sharp band (retinoic acid) or does not migrate at all (retinol). Suspensions of membrane extracts can also be analyzed directly on P A G E without further separation. Protein Transfer, Immunoblotting, and Autoradiography Proteins separated by P A G E are electrophoretically transferred onto a nitrocellulose sheet. Binding proteins can be identified by specific antibodies. The transfer and the binding of proteins to nitrocellulose do not

304

RECEPTORS, TRANSPORT, AND BINDING PROTEINS

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FIG. 4. Direct autoradiography of a nitrocellulose sheet. A human epidermal extract incubated with [3H]retinoic acid was separated on a polyacrylamide gel and transferred to a nitrocellulose sheet. The nitrocellulose sheet was subjected to autoradiography for 1 week with Hyperfilm. In lane 1, the CRABP band and unknown retinoic acid-binding proteins can be detected. Lane 2 shows the disappearance of the radioactive bands in the presence of an excess of unlabeled retinoic acid. constitute denaturing conditions for the retinoid-binding proteins, as indicated b y the fluorescence of the holo-RBP band when the nitrocellulose sheet is e x p o s e d to ultraviolet light (360 nm). This is also confirmed by the profiles obtained w h e n protein extracts incubated with labeled retinoids are separated on P A G E and transferred onto nitrocellulose sheets, before being subjected to a u t o r a d i o g r a p h y with X-ray film (see Fig. 4). L o w a m o u n t s o f retinoid-binding protein can be detected, depending on the duration o f e x p o s u r e time. This p r o c e d u r e increases the resolution, as the gel does not h a v e to be cut into bands. The background is lower, free retinoic acid being r e m o v e d u p o n transfer.

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Tissue and Serum Preparation Skin is one of the most difficult tissues with respect to extraction of proteins. The technique described below has been developed for skin and may also be used for other tissues. Approximately 20 mg of lyophilized human skin is homogenized in an ice bath, in 800/zl of ice-cold extraction buffer [50 mM Tris-HCl, 25 mM NaC1, 2.5 mM EDTA, 1 mM dithiothreitol (DTT), pH 7.5], with three 30-sec strokes of a Polytron PT7 tissue homogenizer (Kinematica, Luzern, Switzerland), at full speed. Supernatants are obtained by centrifugation at 100,000 g at 4° for 60 min, distributed in 100-/xl aliquots, and frozen at - 2 0 ° or used immediately. Fresh or frozen serum with or without delipidation (to remove retinol from RBP) is used as the RBP standard in the PAGE-immunoblotting technique.

Incubation with Retinoids All manipulations are done under dim or yellow light. The supernatants containing 200-300 ~g protein in 100/zl are incubated with 600 nM (saturating conditions for skin extract) of all-trans-[11,12-3H]retinoic acid (50 Ci/mmol, Du Pont, Paris, France) or aU-trans-[11,12-3H]retinol (50 Ci/ mmol, Du Pont). For this purpose, alcoholic solutions of retinoids containing 50/~g/ml of butylated hydroxytoluene as antioxidant are first deposited in glass microtubes, and the solvent is evaporated with a stream of N2. The supernatants are then added, mixed with the pipette, and incubated at 4 ° for 16 hr. For binding studies with ligands of higher affinity, an incubation period of 1 hr at 0° is sufficient. For binding specificity or competition studies, a 200-fold excess of unlabeled ligand is added, in the same manner as for radioactive compounds. At the end of the incubation period an aliquot of the supernatant (90 txl) is subjected to PAGE.

Polyacrylamide Gel Electrophoresis Stock Solutions Acrylamide, 45%: dissolve 56.25 g of acrylamide and 1.5 g N,N'methylenebisacrylamide in 125 ml distilled water and filter the solution Tris-HC1 buffers: 1 M, pH 6.8, and 1 M, pH 8.8 Concentrated (10×) reservoir buffer: dissolve 30.28 g Tris (0.25 M) and 146.38 g glycine in a total volume of 1000 ml of distilled water (final pH 8.8); dilute one-tenth before use Slab Gel Preparation. Quantities are indicated for an LKB-Pharmacia (Uppsala, Sweden) vertical electrophoresis system with two gels (14 cm x 16 cm x 1.5 mm). Each gel contains 10 wells. For the resolving gel, mix

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9.6 ml of Tris-HC1 buffer, pH 8.8, 14.9 ml water, 5.2 ml 45% acrylamide, and 12/xl N,N,N',N'-tetramethylethylenediamine (TEMED). Degas and add 300/zl of a freshly prepared solution of sodium persulfate (30 mg/ml). For the 7.5% stacking gel, mix 1.25 ml of Tris buffer, pH 6.8, 7.35 ml water, 0.64 ml 45% acrylamide, and 5/~1 TEMED. Degas and add 200 ~1 of sodium persulfate solution. Sample Application. Add 20/~1 of a solution containing 0.05% bromphenol blue and 25% glycerol to each sample, in order to facilitate its application into the well. Determination of Radioactivity. After electrophoresis, the gel is divided into lanes and cut into 2-mm bands with a homemade cutter system made of razor blades separated with 2-mm spacers. The bands are collected and treated overnight with 400/zl Protosol (Du Pont) in 5-ml scintillation vials before 4 ml of Pico-fluor (Packard) is added. The radioactivity is then determined in a fl-counter with counting efficiency of 47%. Typical migration profiles are represented in Figs. 2 and 3.

Protein Transfer Proteins separated by PAGE are electrophoretically transferred to nitrocellulose filters at 4 ° in a 0.25 mM sodium phosphate solution at pH 6.5 for 2.5 hr under the electrical conditions recommended by the manufacturers.

Autoradiography of Nitrocellulose Sheets Once the transfer is finished the nitrocellulose sheet is completely dryed with a blower-type dryer. For optimal sensitivity and resolution, the nitrocellulose sheet is placed in close contact with a Hyperfilm (Amersham) in an X-ray cassette. It is important that the side of the film bearing the emulsion is in contact with the nitrocellulose sheet surface, where the proteins have been transferred (see results in Fig. 4). Under these conditions an exposure of 1 week is sufficient for a radioactive band of approximately 5000 dpm.

Immunoblotting of Retinoid Binding Protein After the transfer, the nitrocellulose sheet is treated in a blocking solution for 2 hr [5 mM sodium phosphate, 0.13 mM NaCI, pH 7.2 (PBS), containing 3% defatted powdered milk], prior to a 2-hr incubation at room temperature in PBS containing 0.5% bovine serum albumin, 0.2% Tween 20, and an appropriate amount of goat antiserum directed against human RBP. The filter is then washed 3 times with the incubation solution and

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further incubated for 1 hr at room temperature with horseradish peroxidase-labeled rabbit anti-goat IgG Fab fragment (Cappel, CochranviUe, PA). The filter is then washed 3 times in Tris buffer (100 mM Tris-HCl, pH 7.4) to remove excess second antibody. The bound enzyme is visualized by incubating the sheet in the above Tris buffer containing 0.5 mg/ml diaminobenzidine and 0.03% H202. After the bands appear, the reaction is stopped, and the background coloration is partially removed by replacing the Tris buffer containing the substrates with a solution of 3% acetic acid in water (see Fig. 1). Conclusion The electrophoresis technique enables one to study in detail the extraand intracellular retinoid-binding proteins in human normal skin and in skin diseases affecting keratinization (for review, see Ref. 17). Moreover, this method was used with success to show that terminal differentiation in cultured human keratinocytes was associated with increased levels of CRABP. 14 Acknowledgments This work was supported in part by the Swiss National Science Foundation, Grant 3,874,088. I thank Dr. A. Capponi for rewriting the manuscript. I gratefully acknowledge Raymonde Hotz and Evelyne Leemans for excellent technical assistance and Sylvie Deschamps for typing the manuscript. 17G. Siegenthaler and J.-H. Saurat, Pharrnacol. Ther. 40, 45 (1989).

[31] C e l l u l a r R e t i n o i c A c i d - B i n d i n g P r o t e i n f r o m N e o n a t a l R a t Skin: P u r i f i c a t i o n a n d A n a l y s i s

By CHRISTOPHER P. F. REDFERN and ANN K. DALV Introduction Cellular retinoic acid-binding protein (CRABP) is a member of a family of low molecular weight, hydrophobic ligand-binding proteins which includes cellular retinol-binding protein, myelin protein P2, and fatty acidMETHODS IN ENZYMOLOGY, VOL. 189

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Gel electrophoresis of cellular retinoic acid-binding protein, cellular retinol-binding protein, and serum retinol-binding protein.

[30] PAGE ANALYSISOF CRABP, CRBP, AND RBP 299 foundation for hypotheses and future research concerning the physiological and biochemical roles of e...
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